Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interstitial 3-Methoxytyramine Reflects Striatal Dopamine Release: An In Vivo Microdialysis Study

Previous ex vivo studies have provided indirect evidence that the dopamine (DA) metabolite 3-methoxytyramine (3-MT) may be a useful index of DA release in vivo. In the present study, in vivo microdialysis was utilized to assess directly the relationship between extracellular DA and 3-MT in the striatum of rats following a variety of pharmacological manipulations. Apomorphine, a DA receptor agonist, produced a rapid, transient decrease in both DA and 3-MT. Conversely, the DA receptor antagonist haloperidol produced a concomitant increase in extracellular DA and 3-MT. Increases in DA and 3-MT were also noted following the administration of the DA uptake inhibitor, bupropion. Local application of tetrodotoxin resulted in the complete elimination of measurable amounts of DA and 3-MT in the dialysate, gamma-Butyrolactone also greatly decreased DA and 3-MT. Finally, d-amphetamine produced a large increase in DA and 3-MT in animals that had been treated previously with gamma-butyrolactone. The Pearson correlation coefficients for DA and 3-MT following these manipulations ranged from 0.87 to 0.97. These data indicate that interstitial 3-MT is an accurate index of DA release. However, when compared with previous ex vivo findings, the present results also suggest that changes in tissue concentrations of 3-MT may not reliably reflect DA release following certain pharmacological manipulations.     

Quantitative Microdialysis: Analysis of Transients and Application to Pharmacokinetics in Brain

The behavior of a microdialysis probe in vivo is mathematically described. A diffusion-reaction model is developed that not only accounts for transport of substances through tissues and probe membranes but also accounts for transport across the microvasculature and metabolism. Time-dependent equations are presented both for the effluent microdialysate concentration and for concentration profiles about the probe. The analysis applies either to measuring the tissue pharmacokinetics of drugs administered systemically, or for sampling of endogenously produced substances from tissue. In addition, an expression is developed for the transient concentration about the probe when it is used as an infusion device. All mathematical expressions are found to be a sum of an algebraic and an integral term. Theoretical prediction of time-dependent probe behavior in brain has been compared with experimental data for acetaminophen administered at 15 mg/kg to rats by intravenous bolus. Plasma and whole striatal tissue samples were used to describe plasma kinetics and to estimate a capillary permeability-area product of 0.07 min-1. Theoretical prediction of transient effluent dialysate concentrations exhibited close agreement with experimental data over 60 min. Terminal decline of the dialysate effluent concentration was slightly overestimated but theoretical concentrations still lay within the 95% confidence interval of the experimental data at 112 min. Microvasculature transport and metabolism play major roles in determining microdialysate transient responses. Extraction fraction (recovery) has been shown to be a declining function in time for five probe operating conditions. High rates of metabolism and/or capillary transport affect the time required to approach steady-state extraction, shortening the time as the rates increase. Conversely, for substances characterized by low permeabilities and negligible metabolism, experimental situations exist that are predicted to have very slow approaches to microdialysis steady state.     

The cortisol-cortisone shuttle and hypertension

11β-OHSD is an enzyme complex consisting of 11β-DH, converting cortisol to cortisone in man and an 11-keto-reductase performing the reverse reaction. Congenital deficiency of 11β-DH should be considered in any child presenting with mineralocorticoid hypertension and suppression of the renin-angiotensin-aldosterone axis. The keystone to diagnosis is the demonstration of a reduced daily production rate of cortisol and an increase in its plasma half-life. In the majority of cases diagnosis can be made from a urinary steroid metabolite profile indicating a high excretion of cortisol relative to cortisone metabolites. Cortisol is the responsible mineralocorticoid, and as such treatment with the pure glucocorticoid dexamethasone will prevent life-threatening hypokalaemia, although additional antihypertensive drugs are usually required to control blood pressure.

Liquorice and carbenoxolone, for years thought to be direct “agonists” of the mineralocorticoid receptor, in fact cause sodium retention through inhibition of 11β-DH.

The demonstration of 11β-DH activity in the vasculature raises the possibility that it locally modules access of glucocorticoids to mineralocorticoid and possibly glucocorticoid receptors in the vessel wall.

It remains possible that subtle alterations of this cortisol-cortisone shuttle are responsible for other forms of hypertension which are currently classified under the umbrella diagnosis of essential hypertension.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

The Interactions of Superoxide Ion(O-2) with Metallo-Porphyrins [(C18TPP)M, M = FE,MN,CO,ZN]; Models for Biological Systems and Superoxide Dismutases

In dimethylformamide superoxide ion forms a l:l adduct with tctrakis (2.6-dichlorophenyl) porphinatoiron, (Cl₈ TPP)FeOO^-, as well as with its manganese analogue, (Cl₈ TPP)MnOO^-. On the basis of their electrochemical, spectroscopic, and magnetic properties these adducts have a metal-oxygen covalent bond (PorM-OO^-), oxygen-centered redox chemistry. and reactivities that are similar to the hydroperoxide ion (HOO^-). Addition of ^-OH to a solution of PorFe and O₂ results in the formation of PorFe(OH)(OO^-), which can be electrochemically oxidized to PorFeOH plus O₂ (-0.2 V vs SCE). Addition of protons to the PorM-OO^- adducts promotes their rapid decomposition to PorM, HOOH. and O₂. This chemistry provides insight to the reactions of biological superoxide and superoxide dismutases.     

An activating amino acid substitution in the c-abl oncogene protein fails to produce a local conformational change

Thebcr-abl chimeric gene of Philadelphia chromosome positive chronic myelogenous leukemias is only weakly transforming. This transformation activity is greatly enhanced by a Lys-for-Glu substitution at position 832 in the c-abl gene, as occurs in the highly transforming v-abl genes. It has been suggested that this mutation results in a significant structural change in the encoded protein product. Using conformational energy analysis, we have determined the allowed low-energy conformations for residues 828–836 of this protein with Lys and Glu at position 832. In both cases, the overwhelmingly preferred conformation for this region is a bend-helix motif. The helix terminates at residue 836, and there are no discernible differences in conformation between the Lys- and Glu-containing sequences. These results suggest that the activating amino acid substitution at position 832 in the c-abl protein product does not produce its effect via a local conformational change.     

Estimating ecotoxicological risk and impact using indigenous aquatic microbial communities

Emphasis has increased on accuracy in predicting the effect that anthropogenic stress has on natural ecosystems. Although toxicity tests low in environmental realism, such as standardized single species procedures, have been useful in providing a certain degree of protection to human health and the environment, the accuracy of such tests for predicting the effects of anthropogenic activities on complex ecosystems is questionable. The use of indigenous communities of microorganisms to assess the hazard of toxicants in aquatic ecosystems has many advantages. Theoretical and practical aspects of microbial community tests are discussed, particularly in related to widely cited problems in the use of multispecies test systems for predicting hazard. Further standardization of testing protocols using microbial colonization dynamics is advocated on the basis of previous studies, which have shown these parameters to be useful in assessing risk and impact of hazardous substances in aquatic ecosystems.     

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.